Kinetic stability is a poorly understood property of a select group of naturally occurring proteins that are trapped in their native conformations by an energy barrier, and consequently are resistant to unfolding. Kinetic stability can be best explained by illustrating the unfolding process as a simple equilibrium reaction between two protein conformations, the native folded state (N) and the unfolded state (U), separated by a higher energy transition state (TS) (FIG. 1). Since the height of the TS barrier determines the rate of folding and unfolding, kinetically stable proteins possess an unusually high energy TS, which results in extremely slow unfolding rates that virtually trap the protein in its native state (FIG. 1A). Even though the overall change in Gibbs free energy (ΔG) may be favorable for unfolding under extreme solvent conditions, such as high concentrations of denaturant, the high activation energy of the TS significantly slows down the unfolding rate (FIG. 1B). It has been suggested that the presence of a high kinetic energy barrier separating the folded and unfolded states is an evolutionary feature intended to allow proteins to maintain activity in the extreme conditions they may encounter in vivo (1). The examples of the kinetically stable proteins α-lytic protease (extracellular enzyme) (1), Escherichia coli OmpA (bacterial membrane protein) (2), and pyrrolidone carboxyl peptidase (hyperthermophilic protein) (3), illustrate the kinetic adaptation of proteins that must retain enzymatic function in conditions where degradation might easily take place. In addition, thermodynamic stability alone does not fully protect proteins that are susceptible to irreversible denaturation and aggregation arising from partially denatured states that become transiently populated under physiological conditions (4). Therefore, the development of a high kinetic energy barrier to unfolding may serve to protect susceptible proteins against such harmful conformational “side-effects”.
The physical basis for kinetic stability is poorly understood and no structural consensus has been found to explain this phenomenon. In previous studies, the addition of hydrophobic residues on the protein surface (5), the engineering of disulfide bonds (6), and the introduction of metal-binding sites (7) have been shown to increase kinetic stability. A connection between kinetic stability and oligomeric quaternary structure has also been proposed (8). In the case of some hyperthermophilic proteins, electrostatic interactions have been suggested to be a major factor in their slow unfolding due to the formation of ion pairs (9, 10). However, there is evidence that some kinetically stable proteins retain their slow unfolding rate even at low pH, where electrostatic interactions should be significantly weakened (3, 11). Thus, it appears that no common structural feature exists to explain kinetic stability, and perhaps this property may be achieved by different means, depending on the individual protein.
Under native conditions, kinetically stable proteins have limited access to partially and globally unfolded conformations (12). These properties impart a strong proteolytic resistance by reducing the occurrence of accessible conformations susceptible to proteolytic attack (12, 13). Some kinetically stable proteins have also been found to be resistant to denaturation by sodium dodecyl sulfate (SDS). Among them are the β-sheet proteins streptavidin (14), transthyretin (15), P22 tailspike protein (16), and the e-coli membrane protein, OmpA (2).
The ability to quickly and easily identify kinetically stable proteins would have a myriad of applications in the biotechnology industry, pharmaceutical industry, and in basic life science research.